Light-induced charge generation in polymeric nanoparticles restores vision in advanced-stage retinitis pigmentosa rats.

Retinal dystrophies such as Retinitis pigmentosa are among the most prevalent causes of inherited legal blindness, for which treatments are in demand. Retinal prostheses have been developed to stimulate the inner retinal network that, initially spared by degeneration, deteriorates in the late stages of the disease. We recently reported that conjugated polymer nanoparticles persistently rescue visual activities after a single subretinal injection in the Royal College of Surgeons rat model of Retinitis pigmentosa. Here we demonstrate that conjugated polymer nanoparticles can reinstate physiological signals at the cortical level and visually driven activities when microinjected in 10-months-old Royal College of Surgeons rats bearing fully light-insensitive retinas. The extent of visual restoration positively correlates with the nanoparticle density and hybrid contacts with second-order retinal neurons. The results establish the functional role of organic photovoltaic nanoparticles in restoring visual activities in fully degenerate retinas with intense inner retina rewiring, a stage of the disease in which patients are subjected to prosthetic interventions.

From transformation to chronification of migraine: pathophysiological and clinical aspects.

Chronic migraine is a neurological disorder characterized by 15 or more headache days per month of which at least 8 days show typical migraine features. The process that describes the development from episodic migraine into chronic migraine is commonly referred to as migraine transformation or chronification. Ample studies have attempted to identify factors associated with migraine transformation from different perspectives. Understanding CM as a pathological brain state with trigeminovascular participation where biological changes occur, we have completed a comprehensive review on the clinical, epidemiological, genetic, molecular, structural, functional, physiological and preclinical evidence available.

The role of Trichoderma spp. and silica gel in plant defence mechanisms and insect response in vineyard.

Several elicitors, stimulating induced resistance mechanisms, have potential in preventing or mitigating pathogen infections. Some of these compounds, triggering the production of jasmonic acid (JA), a precursor of herbivore-induced plant volatiles, could also play a central role in indirect resistance to pest species, by improving beneficial arthropod performance, and necrotrophic pathogens. In the current work, Trichoderma gamsii/T. asperellum and silica gel treatments - alone and in combination - were studied to evaluate the plant defence mechanism on grapevines (Vitis vinifera L.) by laboratory and field trials. JA production level was measured before and after Plasmopara viticola infection on potted vines. JA production induced by silica gel was higher than that caused by Trichoderma before infection. In Trichoderma-treated plants, JA production increased after P. viticola inoculation. In vineyard field trials, Mymaridae (Hymenoptera: Chalcidoidea) showed higher captures in transparent sticky traps on silica gel-treated plants, in comparison with control. On the other hand, no significant attraction was detected for Ichneumonoidea and other Chalcidoidea in silica gel and T. gamsii/T. asperellum-treated plants. The potential effects of elicitors are discussed, in the frame of attract and reward strategy.

Inner retinal inhibition shapes the receptive field of retinal ganglion cells in primate.

The centre-surround organisation of receptive fields is a feature of most retinal ganglion cells (RGCs) and is critical for spatial discrimination and contrast detection. Although lateral inhibitory processes are known to be important in generating the receptive field surround, the contribution of each of the two synaptic layers in the primate retina remains unclear. Here we studied the spatial organisation of excitatory and inhibitory synaptic inputs onto ON and OFF ganglion cells in the primate retina. All RGCs showed an increase in excitation in response to stimulus of preferred polarity. Inhibition onto RGCs comprised two types of responses to preferred polarity: some RGCs showed an increase in inhibition whilst others showed removal of tonic inhibition. Excitatory inputs were strongly spatially tuned but inhibitory inputs showed more variable organisation: in some neurons they were as strongly tuned as excitation, and in others inhibitory inputs showed no spatial tuning. We targeted one source of inner retinal inhibition by functionally ablating spiking amacrine cells with bath application of tetrodotoxin (TTX). TTX significantly reduced the spatial tuning of excitatory inputs. In addition, TTX reduced inhibition onto those RGCs where a stimulus of preferred polarity increased inhibition. Reconstruction of the spatial tuning properties by somatic injection of excitatory and inhibitory synaptic conductances verified that TTX-mediated inhibition onto bipolar cells increases the strength of the surround in RGC spiking output. These results indicate that in the primate retina inhibitory mechanisms in the inner plexiform layer sharpen the spatial tuning of ganglion cells.

Subclinical pulmonary edema in endurance athletes.

Strenuous exercise may cause progressive and proportional haemodynamic overload damage to the alveolar membrane, even in athletes. Despite the high incidence of arterial desaturation reported in endurance athletes has been attributed, into other factors, also to the damage of the alveolar-capillary membrane this evidence is equivocal. Some studies demonstrated flood of the interstitial space and consequent increase in pulmonary water content, but most of them were able to show this through indirect signs of interstitial oedema. The present review illustrates the literature's data in favour or against pulmonary interstitial edema due to intense exercise in athletes.

Chromosome painting in two genera of South American monkeys: species identification, conservation, and management.

Cytogenetic studies showed that a number of New World primate taxa, particularly the genera Alouatta, Aotus, and Callicebus, have highly derived karyotypes. Cytogenetics in these primates, at every level of analysis, has contributed to the recognition of species and revealed that their number was certainly underestimated by researchers relying solely on traditional morphological data. Further attention was drawn to Alouatta and Aotus because they are characterized by translocations of the Y chromosome to autosomes, generating multiple sex chromosome systems. Here we present a report on the hybridization of human chromosome-specific paints on metaphases from 4 individuals originally assigned to Alouatta caraya and 1 individual of Aotuslemurinus. This is only the third karyotype studied with chromosome painting out of more than 10 known karyomorphs in Aotus. The banded chromosomes matched those of karyotype II as defined by Ma et al. [1976a], and we were able to more precisely assign the origin of the sample to A. l. griseimembra. Our results on the Argentinean Alouatta caraya samples were generally comparable to the banding and hybridization pattern of previous studies of A. caraya including the presence of an X(1)X(1)X(2)X(2)/X(1)X(2)Y(1)Y(2) sex chromosome system. The karyotype of the Brazilian Alouatta sample labeled as A. caraya differs from the 3 Argentinean samples by at least 10 chromosome rearrangements. The diploid number, G banding, and hybridization pattern of this female cell line was almost identical to previous painting results on Alouatta guariba guariba. Therefore we must conclude that this cell line is actually from an A. guariba guariba individual. The contribution of cytogenetic tools in identifying species or in this case assigning individuals or cell lines to their precise taxonomic allocation is stressed. Gathering further molecular cytogenetic data on New World primates should be conservation and management priorities.

Craniofacial morphometric variation and the biological history of the peopling of Sardinia.

The aim of this work is to explore the pattern of craniofacial morphometric variation and the relationships among five prehistoric Sardinian groups dated from Late Neolithic to the Nuragic Period (Middle and Late Bronze Age), in order to formulate hypotheses on the peopling history of Sardinia. Biological relationships with coeval populations of central peninsular Italy were also analysed to detect influences from and towards extra-Sardinian sources. Furthermore, comparison with samples of contemporary populations from Sardinia and from continental Italy provided an indication of the trend leading to the final part of the peopling history. Finally, Upper Palaeolithic and Mesolithic samples were included in the analyses to compare the prehistoric Sardinians with some of their potential continental ancestors. The analysis is based on multivariate techniques including Mahalanobis D(2) distance, non-parametric multidimensional scaling (MDS) and principal component analysis (PCA). The results showed the tendency to progressive differentiation between Sardinian groups and peninsular Italian groups, with the possible exception of a discontinuity showed by the Bonnànaro (Early Bronze Age) Sardinian sample. Several aspects of the morphological results were found to agree with the current genetic evidence available for the present-day Sardinian population and a Nuragic sample: (1) biological divergence between the Sardinian and peninsular Italian populations; (2) similarity/continuity among Neolithic, Bronze Age and recent Sardinians; (3) biological separation between the Nuragic and Etruscan populations; (4) contribution of a Palaeo-Mesolithic gene pool to the genetic structure of current Sardinians.

Randomized, double-blind, phase III, controlled trial comparing levobupivacaine 0.25%, ropivacaine 0.25% and bupivacaine 0.25% by the caudal route in children.

BACKGROUND: The rationale for replacing racemic bupivacaine with the s-enantiomers levobupivacaine and ropivacaine is to provide a wider margin of safety with the same analgesic efficacy and less postoperative motor block. In a randomized, double-blind, phase III, controlled trial we compared the caudal administration of levobupivacaine 0.25% and ropivacaine 0.25% with bupivacaine 0.25% in children. METHODS: Ninety-nine ASA I-II children less than 10 yr old scheduled for elective sub-umbilical surgery were randomized to receive caudal block with bupivacaine 0.25%, ropivacaine 0.25% or levobupivacaine 0.25%. The primary outcome of the study was the clinical efficacy of the caudal block during the operation. Secondary outcome measures were analgesic onset time, pain relief after the operation and residual motor blockade. RESULTS: The proportion of children with effective analgesia during the operation was similar among groups. There were no significant differences in the analgesic onset time of the caudal block. Bupivacaine produced a significant incidence of residual motor block compared with levobupivacaine or ropivacaine at wake-up (P<0.01). There were no significant differences in the number of patients receiving rescue analgesia after surgery. However, analgesic block lasted significantly longer in patients receiving bupivacaine (P=0.03). CONCLUSION: During sub-umbilical surgery, caudal levobupivacaine, ropivacaine and bupivacaine provided comparable analgesic efficacy. Bupivacaine produced a higher incidence of residual motor blockade and a longer analgesic block than ropivacaine and levobupivacaine.

Effects of heavy ions on visual function and electrophysiology of rodents: the ALTEA-MICE project.

ALTEA-MICE will supplement the ALTEA project on astronauts and provide information on the functional visual impairment possibly induced by heavy ions during prolonged operations in microgravity. Goals of ALTEA-MICE are: (1) to investigate the effects of heavy ions on the visual system of normal and mutant mice with retinal defects; (2) to define reliable experimental conditions for space research; and (3) to develop animal models to study the physiological consequences of space travels on humans. Remotely controlled mouse setup, applied electrophysiological recording methods, remote particle monitoring, and experimental procedures were developed and tested. The project has proved feasible under laboratory-controlled conditions comparable in important aspects to those of astronauts' exposure to particle in space. Experiments are performed at the Brookhaven National Laboratories [BNL] (Upton, NY, USA) and the Gesellschaft für Schwerionenforschung mbH [GSI]/Biophysik (Darmstadt, FRG) to identify possible electrophysiological changes and/or activation of protective mechanisms in response to pulsed radiation. Offline data analyses are in progress and observations are still anecdotal. Electrophysiological changes after pulsed radiation are within the limits of spontaneous variability under anesthesia, with only indirect evidence of possible retinal/cortical responses. Immunostaining showed changes (e.g. increased expression of FGF2 protein in the outer nuclear layer) suggesting a retinal stress reaction to high-energy particles of potential relevance in space.

Clearance of infection with Mycobacterium bovis BCG in mice is enhanced by treatment with S28463 (R-848), and its efficiency depends on expression of wild-type Nramp1 (resistance allele).

The mouse bcg host resistance gene is known to control the activation of host macrophages for killing of intracellular parasites like Leishmania donovani as well as intracellular bacteria, including Mycobacterium bovis BCG and Salmonella enterica serovar Typhimurium. The Nramp1 gene has been mapped to this locus and affects the efficiency of macrophage activation. It has been shown that imidazoquinoline compounds, including S28463, are able to improve the clearance of a number of intracellular pathogens such as herpes simplex virus 2, human papillomavirus, and Leishmania. The goal of this study was to determine whether S28463 is efficient against infection with another intracellular pathogen, M. bovis BCG, and to determine the molecular basis underlying this effect. To achieve this, B10A.Nramp1(r) and B10A.Nramp1(-/-) mice were infected with M. bovis BCG and treated with S28463. The bacterial content in the spleen from these mice was assayed by a colony-forming assay. In addition, in vitro experiments were performed using bone marrow-derived macrophage cell lines from these mice. These cells were treated with S28463 and/or gamma interferon (IFN-gamma), and nitric oxide (NO) production was measured. Our study was able to show that S28463 acts in synergy with IFN-gamma to increase the production of NO in vitro. We were also able to demonstrate that mice that carried the resistant allele of the Nramp1 gene and were infected with M. bovis BCG responded to treatment with S28463, resulting in a decreased bacterial load after 2 weeks of treatment. Mice that do not express the Nramp1 gene responded only to a very large dose of S28463, and the response was not as efficient as that observed in mice carrying a wild-type Nramp1 allele. Our data provide evidence for the potential of S28463 as an immunomodulator that may be helpful in designing efficient strategies to improve host defense against mycobacterial infection.

Polymorphism in the 3'-untranslated region of TNFalpha mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFalpha mRNA.

Tumor necrosis factor alpha (TNFalpha) acts as a beneficial mediator in the process of host defence. In recent years major interest has focused on the AU-rich elements (AREs) present in the 3'-untranslated region (3'-UTR) of TNFalpha mRNA as this region plays a pivotal role in post-transcriptional control of TNFalpha production. Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFalpha mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFalpha. In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3'-UTR of TNFalpha mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFalpha protein. We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFalpha 3'-UTR. One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs. Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFalpha mRNA, these results suggest that mutations in the ARE of TNFalpha mRNA decrease the production of TNFalpha protein in macrophages by hindering the binding of HuR to the ARE.

Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

Inhibition of the hepatitis C virus NS3/4A protease. The crystal structures of two protease-inhibitor complexes.

The hepatitis C virus NS3 protein contains a serine protease domain with a chymotrypsin-like fold, which is a target for development of therapeutics. We report the crystal structures of this domain complexed with NS4A cofactor and with two potent, reversible covalent inhibitors spanning the P1-P4 residues. Both inhibitors bind in an extended backbone conformation, forming an anti-parallel beta-sheet with one enzyme beta-strand. The P1 residue contributes most to the binding energy, whereas P2-P4 side chains are partially solvent exposed. The structures do not show notable rearrangements of the active site upon inhibitor binding. These results are significant for the development of antivirals.

The 1.2 A crystal structure of hirustasin reveals the intrinsic flexibility of a family of highly disulphide-bridged inhibitors.

BACKGROUND: Leech-derived inhibitors have a prominent role in the development of new antithrombotic drugs, because some of them are able to block the blood coagulation cascade. Hirustasin, a serine protease inhibitor from the leech Hirudo medicinalis, binds specifically to tissue kallikrein and possesses structural similarity with antistasin, a potent factor Xa inhibitor from Haementeria officinalis. Although the 2.4 A structure of the hirustasin-kallikrein complex is known, classical methods such as molecular replacement were not successful in solving the structure of free hirustasin. RESULTS: Ab initio real/reciprocal space iteration has been used to solve the structure of free hirustasin using either 1.4 A room temperature data or 1.2 A low temperature diffraction data. The structure was also solved independently from a single pseudo-symmetric gold derivative using maximum likelihood methods. A comparison of the free and complexed structures reveals that binding to kallikrein causes a hinge-bending motion between the two hirustasin subdomains. This movement is accompanied by the isomerisation of a cis proline to the trans conformation and a movement of the P3, P4 and P5 residues so that they can interact with the cognate protease. CONCLUSIONS: The inhibitors from this protein family are fairly flexible despite being highly cross-linked by disulphide bridges. This intrinsic flexibility is necessary to adopt a conformation that is recognised by the protease and to achieve an optimal fit, such observations illustrate the pitfalls of designing inhibitors based on static lock-and-key models. This work illustrates the potential of new methods of structure solution that require less or even no prior phase information.

Multiple determinants influence complex formation of the hepatitis C virus NS3 protease domain with its NS4A cofactor peptide.

The interaction of the hepatitis C virus (HCV) NS3 protease domain with its NS4A cofactor peptide (Pep4AK) was investigated at equilibrium and at pre-steady state under different physicochemical conditions. Equilibrium dissociation constants of the NS3-Pep4AK complex varied by several orders of magnitude depending on buffer additives. Glycerol, NaCl, detergents, and peptide substrates were found to stabilize this interaction. The extent of glycerol-induced stabilization varied in an HCV strain-dependent way with at least one determinant mapping to an NS3-NS4A interaction site. Conformational transitions affecting at least the first 18 amino acids of NS3 were the main energy barriers for both the association and the dissociation reactions of the complex. However, deletion of this N-terminal portion of the protease molecule only slightly influenced equilibrium dissociation constants determined under different physicochemical conditions. Limited proteolysis experiments coupled with mass spectrometric identification of cleavage fragments suggested a high degree of conformational flexibility affecting at least the first 21 residues of NS3. The accessibility of this region of the protease to limited chymotryptic digestion did not significantly change in any condition tested, whereas a significant reduction of chymotryptic cleavages within the NS3 core was detected under conditions of high NS3-Pep4AK complex affinity. We conclude the following: (1) The N-terminus of the NS3 protease that, according to the X-ray crystal structure, makes extensive contacts with the cofactor peptide is highly flexible in solution and contributes only marginally to the thermodynamic stability of the complex. (2) Affinity enhancement is accomplished by several factors through a general stabilization of the fold of the NS3 molecule.

Effect of finasteride in idiopathic hirsutism.

Increased 5 alpha-reductase activity has been found in hair follicles of hirsute women, suggesting a pathogenetic role. The aim of the present study was to evaluate the effect of finasteride in the treatment of idiopathic hirsutism. Twenty-seven women with idiopathic hirsutism, aged 16-35 years, were treated for 6 months with finasteride, 5 mg once daily. Fourteen patients were on finasteride alone (group A), while the remaining received in addition an oral contraceptive (group B). Clinical, hormonal and biochemical evaluation were performed before, and after 3 and 6 months of treatment. Clinical evaluation was repeated 6 months after drug discontinuation in seven patients. Treatment was well tolerated by all patients; no side effects or adverse reactions were reported. A significant improvement of hirsutism was obtained by finasteride; clinical score observed at the 6th month of therapy was reduced from 11.71 +/- 2.23 to 7.92 +/- 1.81 (p < 0.05) and from 14.92 +/- 6.13 to 9.3 +/- 2.75 (p < 0.05) in group A and B, respectively. Clinical score in seven patients was still 8.61 +/- 2.28 (p < 0.05) 6 months after the end of therapy. Finasteride treatment alone (group A) induced a slight increase, though not significant, in serum androgens; DHT and SHBG did not change. In group B (finasteride plus oral contraceptive) total testosterone and free testosterone showed no significant decrease; after 6 months of therapy DHT was reduced significantly, while SHBG levels were increased. These data demonstrate that 5 alpha-reductase inhibition may be an effective treatment in women suffering from idiopathic hirsutism. This approach may be attractive due to the absence of adverse reactions, although the necessity of an adequate contraception should be kept in mind.

Design of selective eglin inhibitors of HCV NS3 proteinase.

Hepatitis C virus (HCV) infection is a major health problem that leads to cirrhosis and hepatocellular carcinoma in a substantial number of infected individuals, estimated to be 100-200 million worldwide. Unfortunately, immunotherapy or other effective treatments for HCV infection are not yet available, and interferon administration has limited efficacy. Different approaches to HCV therapy are being explored, and these include inhibition of the viral proteinase, helicase, and RNA-dependent RNA polymerase and development of a vaccine. Here we present the design of selective inhibitors with nanomolar potencies of HCV NS3 proteinase based on eglin c. These eglin c mutants were generated by reshaping the inhibitor active site-binding loop, and the results emphasize the role played by residues P5-P4' in enzyme recognition. In addition, alanine scanning experiments provide evidence that the N terminus of eglin c also contributes to NS3 binding. These eglin inhibitors offer a unique tool for accurately assessing the requirements for effective inhibition of the enzymatic activity of NS3 and at the same time can be considered lead compounds for the identification of other NS3 inhibitors in targeted design efforts.

Characterization of the RNA binding proteins forming complexes with a novel putative regulatory region in the 3'-UTR of TNF-alpha mRNA.

Tumor necrosis factor-alpha (TNF-alpha) is a key cytokine regulator of an early immune response and the central mediator of deleterious effects of systemic inflammatory response syndrome. High production of TNF-alpha by macrophages requires two signals: the first signal induces transcription, while the second signal releases the translational repression of TNF-alpha mRNA. The translational control of TNF-alpha expression is conferred by sequences in the 3'-untranslated region (3'-UTR) of its mRNA. Previously, we have characterized protein complexes binding to the main AU-rich region in the 3'-UTR of murine TNF-alpha mRNA. Here we describe a second protein binding region which is located 147 bases downstream of the first region and interacts with at least seven distinct protein species present in murine macrophages. The second protein binding motif contains a single AUAUUUAU sequence motif; a mutation of this sequence to AUAGGUAU abrogates the binding of proteins. Some of the macrophage proteins mutually compete for the binding to both regions, while others seem to be region specific. The existence of the two protein binding domains explains the previously published data addressing the translatibility of a reporter gene linked to various deletion mutants of the TNF-alpha 3'-UTR. Both the sequence and position of the two putative protein binding regions are highly conserved across species, indicating their important role in the regulation of translational repression and inducibility of TNF-alpha synthesis.

Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein.

The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed into functional polypeptides by both host- and virus-encoded proteases. The HCV-encoded NS3 protease and its cofactor peptide NS4A form a non-covalent complex, which participates in processing the viral polyprotein. This proteolytic activity is believed to be essential for virus proliferation and thus the NS3 protease is a prime target for developing anti-HCV pharmacological agents. Recent X-ray crystallography structural studies have revealed the nature of this non-covalent complex between NS3 protease and the 'active' central segment of NS4A, providing the opportunity to design a single-chain polypeptide. To this end, the DNA sequence encoding for the NS4A peptide (residues 21-34) was genetically fused via a short linker, capable of making a beta-turn, to the N-terminus of the NS3 protease domain. This engineered single-chain NS3-protease (scNS3) is fully active with kinetic parameters virtually identical with those of the NS3/ NS4A non-covalent complex. Moreover, the scNS3 protease can be displayed on filamentous phage and affinity selected using an immobilized specific inhibitor. The scNS3 expressed as a soluble protein and in a phage-display format facilitates enzyme engineering for further structural studies and in vitro selection of potential drug-resistant mutants. These are important steps towards developing effective anti-protease compounds.